CN104949965B - The background of chromogenic agents solution rises suppressing method, chromogenic agents solution, kit and measurement device - Google Patents
The background of chromogenic agents solution rises suppressing method, chromogenic agents solution, kit and measurement device Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
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- G01N35/1002—Reagent dispensers
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Abstract
The present invention relates to the backgrounds of chromogenic agents solution to rise suppressing method, chromogenic agents solution, kit and measurement device.It provides in keeping for the method when measuring the ingredient in sample, having dissolved the chromogenic agents solution for aoxidizing chromogenic agents for inhibiting the background of the chromogenic agents solution to rise.This method is the measurement of the ingredient for being used in sample to keeping, has dissolved the method that the background rising of the chromogenic agents solution occurred when the chromogenic agents solution for aoxidizing chromogenic agents is inhibited, and has the adjusting process that the hydrogen ion exponent (pH) of the chromogenic agents solution is adjusted to highly acid.Preferably, in the adjusting process, the hydrogen ion exponent of the chromogenic agents solution is adjusted to 2.9 or less or 2.1 or less.Preferably, the chromogenic agents solution is for measuring oxidative stress degree.Preferably, the oxidation chromogenic agents are phenylenediamine derivatives.
Description
Technical field
The present invention relates to for measure the ingredient in sample and dissolved oxidation chromogenic agents chromogenic agents solution back
Scape rises suppressing method, chromogenic agents solution, kit and measurement device.
Background technique
In general, 2% or so of oxygen consumed by vivo is changed into active oxygen radical." active oxygen-free radical "
It is the substance for being both active oxygen and free radical.Although active oxygen-the free radical is due to the work of antioxidant or antioxidase
The biological components such as dynamic active oxygen-free-radical oxidation DNA, lipid, enzyme, the protein for being most eliminated, but not being eliminated.
The oxidative damage of these known biological components is also hard with diabetes, hypertension, artery not only with the hyperfunction association of aging phenomenon
The morbidity association of the diseases such as change.
Herein, the balance of active oxygen-free radical and antioxidant or antioxidase is defined as oxidative stress.Oxidation is answered
Sharp degree is referred to as oxidative stress degree." oxidative stress degree is high " refers to the oxidation as caused by active oxygen-free radical in vivo
Effect and the balance of the antioxidation of antioxidant etc. are destroyed, the hyperfunction situation of oxidation reaction.Oxidative stress degree is high
Situation is bad situation for organism.
The measuring method for measuring oxidative stress degree is described in Patent Documents 1 to 3.The intracorporal lipid of biology, egg
White matter, amino acid, nucleic acid etc., by oxidation reaction, become hydroperoxides (R-OOH) by active oxygen-free radical.To the hydrogen
Peroxide can become the index of oxidative stress degree.In Patent Documents 1 to 3 in documented measuring method, in organism
Active oxygen-free radical of interior generation is not measured directly, and the concentration of the hydroperoxides in resulting blood is made
It is measured with color reaction, the intracorporal oxidative stress degree of biology is evaluated with being integrated into.
In Patent Documents 1 to 3, as the chromogenic agents for above-mentioned color reaction, used as oxidation chromogenic agents
P-phenylene diamine derivative.The p-phenylene diamine derivative has high sensitivity, makes as in hydroperoxide concentration measurement
Chromogenic agents are useful.
However, as described below, there are also amendatory leeway in the conventional art.
That is, as p-phenylene diamine derivative oxidation chromogenic agents it is more unstable under solution state, be easy and
Oxygen reaction in solution, therefore easily cause accidentally color development.Therefore, when being taken care of with solution state, rise there are so-called background
Problem.As a countermeasure, chromogenic agents will be aoxidized by, which carrying out, passes through the freeze-dried method as dried reagent keeping.The drying
Reagent is used by lysate dissolution when in use.Such pre-treatment operation must be carried out when in use to measurement operation
Become burden for person.Accordingly it is desirable to which aoxidizing chromogenic agents can take care of under solution state.
Citation
Patent document
Patent document 1: European Patent No. 0783692B1 specification
Patent document 2: U.S. Patent No. 6,355,489B1 specification
Patent document 3: Japanese Patent Publication 2009-257909 bulletin
Summary of the invention
The present invention be consider the situation and be made, problem of the present invention is that, provide in keeping for sample
In ingredient measurement and dissolved oxidation chromogenic agents chromogenic agents solution when, for inhibiting the back of the chromogenic agents solution
Method that scape rises, chromogenic agents solution, the kit including the chromogenic agents solution and for making for inhibiting background to rise
With the measurement device of the ingredient in the kit measurement sample.
In order to solve the problem above-mentioned, following technological means is taught in the present invention.
By the first aspect of the present invention provide the method for the background of chromogenic agents solution to be inhibited to rise, be used for pair
The background occurred when keeping chromogenic agents solution rises the method inhibited, and the chromogenic agents solution is in sample
The measurement of ingredient, and dissolved oxidation chromogenic agents, which is characterized in that the method has the hydrogen of the chromogenic agents solution
Ion exponent is adjusted to the adjusting process of highly acid.
It preferably, is to constitute as follows, in the adjusting process, the hydrogen ion exponent of the chromogenic agents solution is conditioned
To 2.9 or less or 2.1 or less.
It preferably, is to constitute as follows, the chromogenic agents solution is for measuring oxidative stress degree.
It preferably, is to constitute as follows, the oxidation chromogenic agents are phenylenediamine derivatives.
It preferably, is to constitute as follows, the phenylenediamine derivative is p-phenylene diamine derivative.
Preferably, be to constitute as follows, the p-phenylene diamine derivative by the compound represented by following general formula (I)s or
It is selected in its salt.
Change 1
[in general formula (I), R1、R2、R3And R4Each independently represent hydrogen atom, methyl, ethyl, isopropyl, carbon atom
Hydroxyalkyl, phenyl or the halogen radical of number 1~4.]
By the second aspect of the present invention provide chromogenic agents solution be for the ingredient in sample measurement, dissolved oxygen
Change the chromogenic agents solution of chromogenic agents, which is characterized in that hydrogen ion exponent is adjusted to highly acid.
Preferably, the chromogenic agents solution is to constitute as follows, and hydrogen ion exponent is adjusted to 2.9 or less or 2.1 or less.
Preferably, the chromogenic agents solution is to constitute as follows, for measuring oxidative stress degree.
It preferably, is to constitute as follows, the oxidation chromogenic agents are phenylenediamine derivatives.
It preferably, is to constitute as follows, the phenylenediamine derivative is p-phenylene diamine derivative.
Preferably, be to constitute as follows, the p-phenylene diamine derivative by the compound represented by following general formula (I)s or
It is selected in its salt.
Change 1
[in general formula (I), R1、R2、R3And R4Each independently represent hydrogen atom, methyl, ethyl, isopropyl, carbon atom
Hydroxyalkyl, phenyl or the halogen radical of number 1~4.]
By the kit that the kit that the third aspect of the present invention provides is for the measurement of the ingredient in sample, feature
It is, including the chromogenic agents solution provided by the second aspect of the present invention.
Preferably, the kit is to constitute as follows, further includes the dilution for diluting the chromogenic agents solution, institute
It states dilution and is suitable for the defined hydrogen ion exponent of color reaction by being adjusted to by buffer, the chromogenic agents solution
Hydrogen ion exponent by be changing by the diluted dilution it is described as defined in hydrogen ion exponent.
Measurement device by the fourth aspect of the present invention offer is the measurement device for the measurement of the ingredient in sample,
It is characterized in that, is configured to be measured using the kit provided by the third aspect.
It, can be to for measuring the ingredient in sample, having dissolved the examination of oxidation color development according to a kind of mode of the invention is used
The rising of the background of the chromogenic agents solution of agent is inhibited.As a result, being become with solution state keeping oxidation chromogenic agents
It may.The trouble of dissolution oxidation chromogenic agents is saved when the measurement for carrying out the ingredient in sample as a result, therefore measurement is made
It is convenient for dealer.
Other features and advantages of the invention can be known referring to attached drawing from the explanation of the embodiment of the invention of following progress
Dawn.
Detailed description of the invention
Fig. 1 is schematically shown including the bracket containing chromogenic agents solution according to the present invention and for measuring this
The perspective view of the composition of an example of the measurement system of the measurement device of bracket.
Fig. 2 is the sectional view along the II-II line of measurement system shown in FIG. 1.
Fig. 3 is the oxidation for showing the chromogenic agents solution being filled into the bracket of measurement system shown in FIG. 1 and being dissolved
The figure of the concrete example of chromogenic agents.
Fig. 4 is the flow chart for illustrating an example of movement of measurement system shown in FIG. 1.
Fig. 5 is the chart for showing the confirmation result of chromogenic agents solution (3) of embodiment 2.
Fig. 6 is the chart for showing the confirmation result of chromogenic agents solution (4) of embodiment 2.
Fig. 7 is the chart for showing the confirmation result of chromogenic agents solution (5) of embodiment 2.
Fig. 8 is the chart for showing the confirmation result of chromogenic agents solution (6) of embodiment 2.
Fig. 9 is the chart for showing the confirmation result of chromogenic agents solution (7) of embodiment 2.
Figure 10 is the chart for showing the confirmation result of chromogenic agents solution (8) of embodiment 2.
Figure 11 is the chart for showing the confirmation result of embodiment 3.
Symbol description
A measures system
1 bracket
2 measurement devices
S sample
10 sample slots
11 chromogenic agents solution tanks
12 survey light pond
13 dilution traps
14 dilution liquid baths
15 cleaning liquid baths
At 16 tip fillings
17 tips
21 ozzles
22 survey light unit
23 temperature adjustment blocks
RS1 chromogenic agents solution
RS2 measures liquid
RS3 dilutes sample
RS4 dilution
RS5 cleaning solution
Specific embodiment
Hereinafter, the preferred embodiments of the present invention is concretely demonstrated referring to attached drawing.It is used in the present invention
Numberical range that "~" shows indicate will "~" be before and after documented by the numerical value model that includes respectively as minimum value and maximum value
It encloses.In addition, in the following description, the direction of up and down direction etc. according to attached drawing record.
[measurement system]
Fig. 1 is shown including being applied to chromogenic agents solution of the invention, the bracket containing the chromogenic agents solution and being used for
An example of the measurement system of the measurement device of the oxidative stress degree in organism is measured using the bracket.Measurement system A includes
Bracket 1 and measurement device 2.Bracket 1 is for example arranged in small-sized measurement device 2, for measuring the ingredient contained by sample S
The ward of clinic or hospital is arranged in amount, measurement device 2.As the ingredient in sample S, specifically for example, hydrogen peroxide
Compound (R-OOH).The hydroperoxides are the intracorporal lipid of biology, protein, amino acid, nucleic acid etc. by active oxygen-freedom
Base is aoxidized and is generated.The hydroperoxide concentration obtained by measurement is for the synthetically intracorporal oxidative stress of evaluation biology
Degree.As sample S, specifically, such as the organism samples such as whole blood, blood plasma, serum, urine, saliva, interstitial fluid are enumerated.These examinations
Sample S can be applied like this, can also be diluted with dilution RS4 to apply.
[bracket]
As depicted in figs. 1 and 2, bracket 1 is integrally formed at multiple slots, survey light pond, tip filling etc..1 phase of bracket
When an example of the kit described in the present invention.Bracket 1 for example using polystyrene as material, is manufactured by ejection formation.
Bracket 1 penetrates luminous ray or ultraviolet light in survey light Chi12Chu.Material as bracket 1 as a result, in addition to polystyrene it
Outside, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polycarbonate etc. are also used.Bracket 1 contains
Measure whole reagent required for hydroperoxide concentration.Transport/keeping is refrigerated after the bracket 1 manufacture.As refrigeration item
Part, for example, 4 DEG C or less.
Bracket 1 has sample slot 10, chromogenic agents solution tank 11, surveys light pond 12, dilution trap 13, dilution liquid bath 14, cleaning
16 at liquid bath 15 and tip filling, wherein the content and loading filled are as shown in table 1.It is pasted on the upper surface of bracket 1 18
Sealant (diagram omit).The sealant is made of alumiseal layer, plural layers or film of synthetic resin including aluminium foil,
At least cover chromogenic agents solution tank 11, dilution liquid bath 14 and cleaning liquid bath 15.
Table 1
Sample slot 10 is the slot for dispensing sample S.Sample S is dispensed into using pipette etc. when measurement operator's measurement
Sample slot 10.Sample S is for example by 50 μ L of dispensing.Chromogenic agents solution tank 11 is for filling, takes care of and dissolved the examination of oxidation color development
The slot of the chromogenic agents solution RS1 of agent.In chromogenic agents solution tank 11, such as the chromogenic agents solution filled with 200 μ L
RS1.It surveys light pond 12 and accommodates the measurement liquid RS2 for being mixed with chromogenic agents solution RS1 and sample S, be measurement by oxidation oxidation color development
The reaction of reagent and the part of colour generation generated.Dilution trap 13 dilutes sample S by dilution RS4, is for generating dilution
The slot of sample RS3.Dilution liquid bath 14 is the slot for filling in order to dilute the dilution RS4 that sample S is used.Liquid bath 14 is diluted to make
It is functioned for the container for taking care of dilution RS4.In dilution liquid bath 14, such as fill 400 μ L dilution RS4.Cleaning
Liquid bath 15 is the slot for filling the cleaning solution RS5 for cleaning tip 17.In cleaning liquid bath 15, such as to fill 400 μ L clear
Washing lotion RS5.Tip 17 is used to shift sample S, chromogenic agents solution RS1, dilution RS4 from the slot being filled to other slots,
Or it is stirred when mixing these liquid.16 be the place for loading, keeping tip 17 at the filling of tip.16 at the filling of tip
Can also be used as waste liquid tank, discard should not liquid.
[chromogenic agents solution]
The chromogenic agents solution RS1 being filled in chromogenic agents solution tank 11 is that oxidation chromogenic agents are dissolved in a solvent
Liquid reagent.Such as use water as solvent.In chromogenic agents solution RS1, the concentration for aoxidizing chromogenic agents is adjusted to
It is finally 0.2~8mM or 0.4~6.4mM in measurement liquid RS2.Chromogenic agents are aoxidized to pass through if being oxidized in the molecule
It generates chromophore and carrys out colour generation.Therefore, the concentration of chromogenic agents is aoxidized in chromogenic agents solution RS1, such as is adjusted to
0.24mM~9.6mM or 0.48mM~7.68mM.Oxidation chromogenic agents by be oxidized into half quinochrome or quinochrome to
Color development.As oxidation chromogenic agents, phenylenediamine derivative is used.In particular it is preferred to use p-phenylene diamine derivative.P-phenylenediamine
Derivative is especially the compound or its salt indicated with following general expressions (I).
Change 1
In general formula (I), R1、R2、R3And R4It is separately hydrogen atom, methyl, ethyl, isopropyl, carbon atom number
1~4 hydroxyalkyl, phenyl or halogen radical.As the hydroxyalkyl of carbon atom number 1~4, for example, methylol, ethoxy,
Hydroxypropyl.As the example of ethoxy, 2- ethoxy can be enumerated.
Particularly, in general formula (I), R1、R2、R3And R4Among at least one be methyl, ethyl, isopropyl, carbon atom
Hydroxyalkyl, phenyl or the halogen radical of number 1~4.
Alternatively, R1、R2、R3And R4Among 2 or more be separately methyl, ethyl, isopropyl, carbon atom number 1~4
Hydroxyalkyl, phenyl or halogen radical.Particularly, in R1、R2、R3And R4Among 2 or more separately indicate isopropyl, carbon
In the case where the hydroxyalkyl or phenyl of atomicity 1~4, can be identical group is 2 or more, is also possible to each different 2
A above group.
Alternatively, R1、R2、R3And R4Among at least one be phenyl, and R1、R2、R3And R4Among at least one be carbon atom number 1
~4 hydroxyalkyl.In this case, in the compound indicated with general formula (I), phenyl in conjunction with nitrogen-atoms and the hydroxyl
The nitrogen-atoms that alkyl combines can be the same or different.
Alternatively, the p-phenylene diamine derivative indicated with general formula (I) is being that have above-mentioned substituent R1~R4Among phenyl
Substituent group in addition is as R1~R4Compound in the case where, be also possible to the salt of the compound indicated with general formula (I).Make
For the example of the salt, hydrochloride, sulfate, oxalates or acetate etc. can be enumerated.
Alternatively, p-phenylene diamine derivative is the R in general formula (I)1It is hydrogen atom, R2It is hydrogen atom or isopropyl, R3It is
The hydroxyalkyl of hydrogen atom, ethyl or carbon atom number 1~4, R4It is the hydroxyalkyl of carbon atom number 1~4 or the compound of phenyl.At this
In the case of, it include R in general formula (I)1It is hydrogen atom, R2It is hydrogen atom, R3It is the hydroxyalkyl of ethyl or carbon atom number 1~4, R4
It is hydrochloride, sulfate, oxalates or the acetate etc. of the compound of the hydroxyalkyl of carbon atom number 1~4.
As described above, including R in general formula (I) in p-phenylene diamine derivative1、R2、R3And R4Among at least one be carbon
The compound of the hydroxyalkyl of atomicity 1~4.R1、R2、R3And R4Among at least one be carbon atom number 1~4 hydroxyalkyl to benzene
Diamine derivative shows sharp absorption as oxidative stress degree measurement reagent with high sensitivity.Thus, it is possible to right by this
Phenylenediamine derivative carries out the oxidative stress degree measurement of higher precision.
In addition, as described above, being included in general formula (I) in p-phenylene diamine derivative, R1、R2、R3And R4Among at least
1 be phenyl compound.In the R of p-phenylene diamine derivative1、R2、R3And R4Among in the case that at least one is phenyl, although
Due to its molecular configuration, there are the similarities and differences, but by oxidation and the pigment of colour generation is in the wavelength zone of the wide scope of 420nm~800nm
There is absorbing wavelength in domain.
Thus, for example being existed in the case where the organism sample measured as oxidative stress degree uses blood by selection
The wavelength region for the long wavelength side that interference is not influenced caused by by the presence as blood constituents such as hemoglobins is (for example, 600nm
Above region) in also with the above-mentioned p-phenylene diamine derivative of absorbing wavelength, the measurement of high-precision oxidative stress degree becomes
It may.
The case where changing, is generated according to type of measurement device 2 etc. in addition, though there are measurement sensitivities, but this is right
Phenylenediamine derivative has absorbing wavelength in the wavelength region of wide scope, therefore exists and be difficult to be caused by by measurement device 2
Sensitivity change the advantages of.Particularly, use LED as measurement light source in the case where, although in the presence of by LED batch between
The case where difference causes measurement sensitivity to change, but even in this case, by using the p-phenylene diamine derivative, essence
High oxidative stress degree measurement is spent also to be possibly realized.
As the oxidation chromogenic agents in the present invention, the case where having used the salt of the compound indicated with general formula (I)
Under, improving when due to modulation chromogenic agents solution to the dissolubility of solvent is convenient in manufacture.
The concrete example of the p-phenylene diamine derivative as oxidation chromogenic agents is shown in FIG. 3.Chemical formula (II)~(X) points
Not Biao Shi N, N- dimethyl-Isosorbide-5-Nitrae-phenylenediamine (II), N- ethyl-N- (2- ethoxy)-Isosorbide-5-Nitrae-phenylenediamine (III), N, the bis- (2- of N-
Ethoxy)-Isosorbide-5-Nitrae-phenylenediamine (IV), N- isopropyl-N '-phenyl-Isosorbide-5-Nitrae-phenylenediamine (V), N- phenyl-Isosorbide-5-Nitrae-phenylenediamine (VI),
N- isopropyl-N- phenyl-Isosorbide-5-Nitrae-phenylenediamine (VII), N- ethyl-N- (2- ethoxy)-Isosorbide-5-Nitrae-phenylenediamine sulphate (VIII), N,
Bis- (the 2- ethoxy)-Isosorbide-5-Nitrae-phenylenediamine sulphates (IX) of N-, N, N- dimethyl-Isosorbide-5-Nitrae-phenylenediamine sulphate (X).But this hair
Oxidation chromogenic agents in bright are not limited as these.
Chromogenic agents solution RS1 is adjusted to hydrogen ion exponent (pH) condition into highly acid.Chromogenic agents solution RS1 passes through
Addition strong acid adjusts hydrogen ion exponent to become highly acid.As strong acid, appointing in inorganic acid and organic acid can be used
It is a kind of.As inorganic acid, for example, sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid etc..As organic acid, for example,
Lactic acid, acetic acid, propionic acid, p-methyl benzenesulfonic acid, glycine, phthalic acid, maleic acid, citric acid, succinic acid, oxalic acid, tartaric acid,
Acetic acid etc..It is used alternatively, it is also possible to mix them.
In chromogenic agents solution RS1, hydrogen ion exponent is adjusted to highly acid.In the present invention, highly acid is, for example,
Lower than pH3.0.Specifically, in chromogenic agents solution RS1, hydrogen ion exponent be adjusted to pH2.9 or less (pH0~pH2.9),
PH2.5 or less (pH0~pH2.5), pH2.1 or less (pH0~pH2.1), pH2.0 or less (pH0~pH2.0), pH1.0~
PH2.9, pH1.0~pH2.5, pH1.0~pH2.1, pH1.0~pH2.0 are lower than pH1.0.Chromogenic agents solution RS1 is by one side
Verification hydrogen ion exponent adds strong acid on one side to adjust.In the present invention, when adjusting chromogenic agents solution RS1, Ke Yixian
So that hydrogen ion exponent condition is become highly acid and be redissolved oxidation chromogenic agents, can also first dissolve oxidation color development in water and other solvents
Hydrogen ion exponent condition is adjusted to highly acid again by reagent.
Chromogenic agents solution RS1 can be adjusted to highly acid by making strong acid concentration become defined concentration.In the feelings
Under condition, the concentration of inorganic acid or organic acid be for example added to 0.1M or more, 0.0001mM~1.0M, 0.01mM~1.0M and
Either one or two of 0.1M~1.0M.
It adjusts the chromogenic agents solution RS1 that finishes and is for example filled into Fig. 1 and bracket shown in Fig. 21 by 200 μ L every time
In chromogenic agents solution tank 11.
[dilution]
Dilution RS4 is for diluting sample S.In addition, dilution RS4 is for diluting chromogenic agents solution RS1.
When sample S is diluted liquid RS4 dilution, dilution sample RS3 is generated.Dilution sample RS3 is mixed with chromogenic agents solution RS1,
Generate measurement liquid RS2.Thus, the result is that dilution RS4 dilutes chromogenic agents solution RS1.
As dilution RS4, such as by from acetate buffer solution, phosphate buffer, TRIS- maleate buffer, lemon
Acid buffer, 2- (N- morpholinyl) ethanesulfonic acid (MES) buffer, N- (2- acetylamino) iminodiacetic acid (ADA) buffer
With select to use in BIS-TRIS buffer etc..In these buffers, preferably acetate buffer solution, MES buffer or TRIS- horse
Come acid buffer, more preferable acetate buffer solution.
The hydrogen ion exponent of the dilution RS4 is adjusted in the range of pH3.6~pH5.6 or pH4.6~pH5.4.
The concentration of buffer contained by dilution RS4 is adjusted to eventually become 100mM~400mM or 200mM in measurement liquid RS2
~400mM.Therefore, the concentration of buffer is in dilution RS4, for example, be adjusted to 667mM~2667mM or 1333mM~
2667mM.The hydrogen ion exponent of chromogenic agents solution RS1 is made when being diluted liquid RS4 dilution by the buffering of the buffer
With the hydrogen ion exponent for being changing into dilution RS4.
Adjust the dilution that the dilution RS4 finished is for example filled into Fig. 1 and bracket shown in Fig. 21 by 400 μ L every time
In slot 14.
Chromogenic agents solution RS1 or dilution RS4 includes transition metal salt.The transition metal salt is in order to make by sample S institute
Hydroperoxides group (R-OOH) generation contained is added by the free radical that alkoxy (R-O) and peroxy (R-OO) are constituted.
As the transition metal salt, for example, ferric sulfate (II) (chemical formula FeSO4), copper sulphate (I) (chemical formula Cu2SO4)
Deng.Among this, ferric sulfate (II) is more preferable.The concentration of chromogenic agents solution RS1 or the transition metal salt in dilution RS4 is adjusted
Section is in measurement liquid RS2 finally in the range of 0.0005mM~0.06mM or 0.001mM~0.03mM.Therefore, the transition
Metal salt in the case where being included in chromogenic agents solution RS1, such as be adjusted in 0.0006mM~0.072mM or
The range of 0.0012mM~0.036mM.On the other hand, the transition metal salt is in the case where being included in dilution RS4,
Such as it is adjusted to the range in 0.003mM~0.4mM or 0.007mM~0.2mM.
[tip]
Tip 17 as depicted in figs. 1 and 2, when the preparation for measuring oxidative stress degree, is installed in measurement device 2
The front end of ozzle 21 (aftermentioned) is taken out from tip filling place 16.Tip 17 has the sampling pump unit by measurement device 2
The movement of 21A and draw, the function of drain sample S or chromogenic agents solution RS1 etc..Tip 17 is used in bracket 1 as a result,
The transfer of each liquid or the stirring carried out by absorption/discharge.Tip 17 is used the synthetic resin systems such as polypropylene or polyethylene
It makes.
[measurement device]
Measurement device 2 is for using bracket 1 to measure the intracorporal oxidative stress degree of biology.As depicted in figs. 1 and 2, it surveys
Determine device 2 to include control unit 20, ozzle 21, survey light unit 22 and temperature adjustment block 23.
Control unit 20 is the movement processing for controlling measurement device 2.As shown in Figure 1, control unit 20 is via control line
24 connect with pump unit 21a, ozzle driving unit 21b, survey light unit 22, temperature adjustment block 23 etc. is sampled.Control unit 20 have CPU and
Memory.Interface circuit is configured between constituent elements and the CPU as needed in above-mentioned sampling pump pond 21a etc..It is described
The movement that CPU controls measurement device 2 by executing the program that the memory is stored is handled.The memory includes ROM
Region and ram region.The ROM area accommodates movement processing routine, parameter, standard curve of measurement device 2 etc..The RAM
Region temporarily stores the data obtained by survey light unit 22 other than movement processing routine etc..Control unit 20 is based on the data
Calculate absorbance.Control unit 20 is calculated in sample S using the standard curve that the ROM area is stored based on the absorbance
Hydroperoxide concentration, and calculate oxidative stress degree.In addition, control unit 20 is configured to also directly display the extinction
Degree.
As shown in Figure 1, ozzle 21 is configured to be acted by sampling pump unit 21a and ozzle driving unit 21b.
Control unit 20 controls the movement of ozzle 21 by the movement of control sampling pump unit 21a and ozzle driving unit 21b.
Ozzle 21 front end install tip 17, for will from bracket 1 slot draw liquid be dispensed to other slots or
By the liquid being mixed with by drawing/ejecting mixing.Ozzle 21, which also has, opens bracket 1 set in measurement device 2
Upper surface 18 close to sealant (diagram omit) function.Ozzle 21 such as with stainless steel formed, to break through the sealing
Layer and the mode of opening hole is configured.
On slot required for measuring in oxidative stress degree or the opening portion for surveying light pond etc. after aperture, ozzle 21 is by its front end
Portion is inserted into 16 tip 17 loaded at the filling of tip, extracts tip 17 from tip filling place 16.Tip 17 is mounted as a result,
Onto ozzle 21.Ozzle 21 is configured to show on the upper surface of bracket 1 18 with arrow N1 by ozzle driving unit 21b
Direction out is moved horizontally to destination locations, and moves up and down with the direction shown in arrow N2.It is mounted with the pipe at tip 17
Mouth 21 is moved to the position of purpose slot, and the front end at tip 17 is made to reach the liquid level in the slot by declining.Ozzle 17 is logical
The movement of over sampling pump unit 21a is by slot or surveys in the liquid assimilating to tip 17 in light pond.Thereafter, ozzle 21 is moved to work
For the purpose of other slots or survey light pond, by the liquid absorbed in tip 17 to they dispense and shift.
Survey the colour generation for the measurement liquid RS2 that light unit 22 is used to survey in light pond 12.As shown in Figure 1, surveying light unit 22 has hair
Light portion 22a and acceptance part 22b.Illumination region 22a and acceptance part 22b is when bracket 1 is arranged in measurement device 2 by across survey light
Pond 12 is opposed to configure.Illumination region 22a has LED (diagram omit), such as by the light of 510nm to the direction shown in arrow N3
Irradiation.On the other hand, acceptance part 22b has photodiode (diagram is omitted), receives to issue from illumination region 22a, has passed through survey
The light in light pond 12.Such luminous, light the control surveyed in light unit 22 is carried out by control unit 20.
Temperature adjustment block 23 is for bracket 1 to be adjusted the temperature to the measurement suitable for oxidative stress degree.In temperature adjustment block 23
Equipped with heater (diagram is omitted).Control unit 20 is existed by controlling unlatching/closing of the heater to control the temperature of bracket 1
Defined range.Control unit 20 for example controls bracket 1 at 37 DEG C.
[measurement of oxidative stress degree]
Herein, illustrate the survey for carrying out oxidative stress degree using bracket 1 and measurement device 2 by flow chart shown in Fig. 4
An example of fixed sequence.The movement of measurement device 2 is controlled by control unit 20.In the d-ROMS test of measurement oxidative stress degree
In (Reactive Oxygen Metabolites, active oxygen metabolic product), be not direct measurement organism active oxygen-from
By base.Resulting hydroperoxides (R-OOH) concentration is used color reaction to measure, the intracorporal oxidative stress degree quilt of biology
Synthetically evaluate.Because hydroperoxides (R-OOH) are to receive lipid, the egg of oxidation reaction by active oxygen-free radical
The general name of white matter, amino acid, nucleic acid etc. becomes the index of oxidative stress degree.
As shown in Fig. 2, being separately filled in the chromogenic agents solution tank 11, dilution liquid bath 14, cleaning liquid bath 15 of bracket 1
Chromogenic agents solution RS1, dilution RS4, the cleaning solution RS5 of specified amount.Operator is measured for example using pipette etc. by sample
50 μ L are dispensed into sample slot 10.In this embodiment, as sample S for example using blood plasma.Then, operator is measured to measurement device
2 setting brackets 1.Then, measurement operator closes the lid (diagram is omitted) of measurement device 2 and presses start button and (illustrates and save
Slightly).Hereafter, measurement device 2 automatically acts, and measures oxidative stress degree.
Ozzle 21 declines as shown by arrows in Fig. 2, and the tip 17 that at the filling of tip 16 are loaded is installed to front end
(S1).The ozzle 21 at tip 17 is mounted with to move between each slot shown in arrow N1, in each slot as shown in arrow N2 on move down
It is dynamic.It is mounted with that the ozzle 21 at tip 17 is moved to dilution liquid bath 14 along bracket 1, draws dilution RS4.Thereafter, ozzle 21 moves
Dilution trap 13 is moved, the dilution RS4 of 126 μ L is dispensed into dilution trap 13 (S2).Thereafter, ozzle 21 is moved to sample slot 10,
It is mobile to dilution trap 13 after having drawn sample S, the sample of 14 μ L is dispensed into (S3) to dilution trap 13.Thereafter, ozzle 21 to
The movement of liquid bath 15 is cleaned, draws or discharge cleaning solution RS5 is to clean the front end and inside (S4) at tip 17.
Dilution RS4 is acetate buffer solution, and hydrogen ion exponent is adjusted to pH5.0.The hydrogen ion concentration quilt of sample as a result,
Stablize.Also, in dilution RS4, by measurement liquid RS2 in finally for 0.03mM in a manner of contain ferric sulfate (II)
(FeSO4) it is used as transition metal salt.Therefore, the concentration of the transition metal salt in dilution RS4 is for example adjusted to 0.2mM.By
In the effect of transition metal salt, the hydroperoxides group (R- by being formed during oxidation in organism is generated
OOH), the free radical being made of alkoxy (R-O) and peroxy (R-OO).
Then, ozzle 21 is moved to the position of chromogenic agents solution tank 11, after having drawn chromogenic agents solution RS1,
The chromogenic agents solution RS1 of 150 μ L is dispensed into (S5) to light pond 12 is surveyed.Then, ozzle 21 is moved to dilution trap 13, is drawing
After diluting sample RS3, it is moved to the position for surveying light pond 12, the dilution sample RS3 of 30 μ L is dispensed into (S6) to light pond 12 is surveyed.By
This, the liquid measure of measurement liquid RS2 becomes 180 μ L.Ozzle 21 is molten by the chromogenic agents drawn or discharge is mixed in survey light pond 12
Liquid RS1 is stirred (S7) with the measurement liquid RS2 for diluting sample RS3.Temperature adjustment block 23 will survey light pond 12 and keep the temperature 5 points with 37 DEG C
Clock.Thereafter, the absorbance (S8) that light unit 22 measures 510nm is surveyed.Control unit 20 carrys out base using the standard curve being previously set
Hydroperoxide concentration (S9) is calculated in the absorbance.Control unit 20 is based ultimately upon the hydroperoxide concentration and calculates oxidation
It stress spend.
In chromogenic agents solution RS1, bis- (2- the ethoxy)-Isosorbide-5-Nitraes of N, N--phenylenediamine sulphate is as oxidation chromogenic agents
It is dissolved, such as by addition sulfuric acid at highly acid.At the time of the dilution sample RS3 containing acetate buffer solution is added, survey
The hydrogen ion exponent for determining liquid RS2 is changing into pH5.0 suitable for color reaction.The oxidation chromogenic agents are in surveying light pond 12 if connect
Touching free radical is then oxidized, and is melted into radical cation according to the quantitative change of free radical, is in reddish violet.The intensity of the colour generation of reddish violet
The concentration of hydroperoxides of the reflection in blood, with the cell influenced by active oxygen-free radical, as the by-product of molecule
The amount of the reactive oxygen metabolite (ROMS) of object is directly proportional.
In the case where measuring whole blood as sample S, it is able to use the R of general formula (I)1、R2、R3And R4Among at least one
It is the compound of phenyl as oxidation chromogenic agents.As described above, being in by oxidation in the oxidation chromogenic agents with the construction
In the case where color, the pigment in the wavelength region of the wide scope of 420nm~800nm with absorbing wavelength is generated.Thereby, it is possible to
Measurement wavelength to avoid the absorption of hemoglobin is measured.As the measurement wavelength, for example, by using the wave of 600nm or more
It is long.
As described above, according to the present embodiment, chromogenic agents solution RS1 is at highly acid.Make as a result, dissolved to benzene two
The oxidation chromogenic agents of amine derivative etc. are stablized, therefore are able to suppress the chromogenic agents solution that oxidation chromogenic agents have been dissolved
The rising of the background of RS1.At this moment, the reactivity for aoxidizing chromogenic agents is maintained without being deteriorated.Thus, oxidation chromogenic agents are made
It is possibly realized for liquid reagent adjusting/transport/keeping.Eliminating as a result, will oxidation color development examination before measuring oxidative stress degree
Agent is dissolved into the trouble in solvent, therefore convenient for measurement operator.In addition, the good measurement of precision can be quickly got
Data.
When using apparatus for automatically measuring in the assay, color development will be aoxidized for dry before measuring oxidative stress degree by existing
Reagent is dissolved into the case where way that the movement in water equal solvent is executed as the apparatus for automatically measuring.In such automatic survey
Determine in device, being sufficiently carried out dry the re-dissolving for oxidation chromogenic agents becomes an important factor for ensuring measurement accuracy.
However, there are the dissolutions of the dry oxidation chromogenic agents not to be sufficiently carried out by the apparatus for automatically measuring, therefore measure
The case where precision deteriorates.In contrast, according to the present embodiment, the dry oxidation color development examination carried out by measurement device 2 is not needed
The dissolution of agent, therefore the burden of measurement device 2 tails off.The reduction of the manufacturing cost of measurement device 2 can be brought.In addition, due to
The chromogenic agents solution RS1 that can be directly completely dissolved using oxidation chromogenic agents, therefore measurement accuracy can be remained
Good state.Thus, the chromogenic agents solution RS1 of present embodiment is suitable for the survey in the measurement for automatically carrying out oxidative stress degree
Determine to use in device 2.
In manufacturing the dried reagents such as freeze-dried reagent, needs precisely to dispense and dissolved oxidation chromogenic agents
The dispensing equipment of lysate, or need freeze-drying machinery.According to the present embodiment, since oxidation chromogenic agents can be made
For liquid reagent adjusting/transport/keeping, therefore when manufacture oxidative stress degree measurement is with bracket 1, do not need particularly to dispense
Equipment or drying equipment.Thereby, it is possible to seek the reduction of the manufacturing cost of oxidative stress degree measurement bracket 1.
Bracket 1 is also adjusted containing the dilution RS4 for diluting chromogenic agents solution RS1, dilution RS4 by buffer
Section is the defined hydrogen ion exponent suitable for color reaction.Be adjusted to as a result, the hydrogen of the chromogenic agents solution RS1 of highly acid from
Subindex is changing into the hydrogen ion exponent of dilution RS4 and being diluted liquid RS4 dilution.Thereby, it is possible to precisely survey
Determine oxidative stress degree.
The present invention is not limited as the content of above-mentioned embodiment.The background of chromogenic agents solution according to the present invention
The specific composition of technique of rising suppressing method can carry out various freely changing.Similarly, color development according to the present invention
Reagent solution, kit and the specific composition using the measurement device of the kit can carry out various free design alterations.
In the present invention, kit is not limited to have multiple slots or surveys the bracket 1 in light pond.Being also included in kit will fill out
That the container and transition metal salt for having filled the chromogenic agents solution for the highly acid that oxidation chromogenic agents have been dissolved have been dissolved,
It is filled with the matched device of container of the dilution RS4 for diluting sample S.
In addition, in the present invention, measurement device is not limited to the measurement device 2 of measurement bracket 1.It also include drawing to try from bottle
The reagent is dispensed to survey light pond seperated with bottle and preparing, and measures the measurement device of the variation of colour generation by agent.
In addition, in the present invention, oxidation chromogenic agents are not limited to phenylenediamine derivative.Aoxidizing chromogenic agents also includes tetramethyl
The benzidine derivatives such as base benzidine or o-tolidine.In addition, also including Te Linde reagent or pheno thiophene in oxidation chromogenic agents
Oxazine derivatives.
Embodiment
Hereinafter, specifically describing effect of the invention based on embodiment.Either one or two of the present invention is not limited to these Examples.
[embodiment 1]
It has been modulated according to prescription shown in table 2 and has compared chromogenic agents solution (1), (2), chromogenic agents solution (1), (2).Hair
Color reagent solution (1), (2) are by addition sulfuric acid into highly acid.As oxidation chromogenic agents, using N, bis- (the 2- hydroxyl second of N-
Base)-Isosorbide-5-Nitrae-phenylenediamine sulphate or N- ethyl-N- (2- ethoxy)-Isosorbide-5-Nitrae-phenylenediamine sulphate.Chromogenic agents solution (1),
(2) sulfuric acid concentration in is 0.1M.Compare chromogenic agents solution (1), (2) other than not adding sulfuric acid with chromogenic agents solution
(1), (2) are identical composition.
After the coloring after the adjusting that confirmed each reagent solution, 40 DEG C of keepings keeping in 14 days, visually to compare background
The degree of coloring.Coloring more strong representation background rises bigger.
Table 2 (prescription of embodiment 1)
As shown in table 3, after adjustment, compare chromogenic agents solution (1), (2), chromogenic agents solution (1), (2) are all
Bright, without difference.After 40 DEG C have been taken care of 14 days, compare chromogenic agents solution (1), (2) all consumingly colour purple.Separately
On the one hand, chromogenic agents solution (1), (2) are slightly pink colour or transparent respectively.Know to take care of under highly acid as a result, molten
The rising of the background of liquid is suppressed.
Table 3 (the confirmation result of embodiment 1)
[embodiment 2]
It is adjusted according to prescription shown in table 4 and compares chromogenic agents solution (3) and chromogenic agents solution (3)~(8).As
Aoxidize chromogenic agents, using N, bis- (the 2- ethoxy)-Isosorbide-5-Nitrae-phenylenediamine sulphates of N-.The concentration for aoxidizing chromogenic agents is 3.2mM.
As strong acid, sulfuric acid, hydrochloric acid, lactic acid, acetic acid, propionic acid, p-methyl benzenesulfonic acid are used.Each color development is tried by adding various strong acid
The hydrogen ion exponent of agent solution is adjusted to appearance shown in Fig. 7.Each chromogenic agents solution is taken care of with 40 DEG C of condition, after adjusting,
On day 3, the coloring of the 7th day measurement background.Measurement is proceed as follows, and chromogenic agents solution (3)~(8) are dispensed into branch respectively
The survey light pond 12 of frame 1, sets measurement device 2 in a manner of it can show the absorbance of 510nm.Compare chromogenic agents solution (3)
Measurement with other than not adding strong acid with condition identical with chromogenic agents solution (3)~(8) progress.
Table 4 (prescription of embodiment 2)
Aoxidize chromogenic agents: bis- (the 2- ethoxy)-Isosorbide-5-Nitrae-phenylenediamine sulphates of N, N-
Result is shown in Fig. 5~Figure 10.Fig. 5~Figure 10 is chromogenic agents solution (3)~(8) respectively and compares chromogenic agents
The comparison result of solution (3).In each chart, the longitudinal axis indicates the absorbance at 510nm, and horizontal axis indicates in 40 DEG C of keeping day
Number.The variation of absorbance is bigger, and it is bigger to indicate that background rises.The chart of each chromogenic agents solution is unrelated with the type of strong acid to be shown
Same tendency.I.e., it was found that following tendency, the low maintaining requirement of hydrogen ion exponent more makes in background compared with the type of strong acid
The degree risen becomes smaller.
Bracket of the invention is using the preservation of Leng KURA condition (4 DEG C or less) as premise.Yi Leng KURA carry out preservation with
40 DEG C of preservation is compared in advance in respect of absolute good storage stability.In the case where imaginary Leng KURA saves, sent out from compared with
The comparison of color reagent solution (3) confirmed if it is compare it is short-term between, can inhibit under the conditions of pH2.9 is below background
Color.In addition, it is thus identified that if it is pH2.1 condition below, even if prolonged save, can also background be inhibited to rise.
[embodiment 3]
It is had adjusted according to prescription shown in table 5 and compares chromogenic agents solution (4) and chromogenic agents solution (9).As oxidation
Chromogenic agents, using N, bis- (the 2- ethoxy)-Isosorbide-5-Nitrae-phenylenediamine sulphates of N-.In chromogenic agents solution (9), oxidation color development examination
The concentration of agent is 3.2mM.As strong acid, p-methyl benzenesulfonic acid is used.The concentration tune of p-methyl benzenesulfonic acid in chromogenic agents solution (9)
Section is 0.1M.Comparing chromogenic agents solution (4) other than not adding strong acid is the same terms with chromogenic agents solution (9).
Table 5 (prescription of embodiment 3)
Chromogenic agents solution (4) and chromogenic agents solution (9) 14 days are compared in 40 DEG C of keepings, compare after adjusting and after keeping
Reactivity.
When measurement, as sample S, the aqueous solution of tert-butyl hydroperoxide (t-BuOOH) has been used.Tert-butyl hydrogen mistake
Oxide is used as the standard substance of hydroperoxides (R-OOH).Tert-butyl hydroperoxide solution is adjusted to final measurement liquid
Concentration (quality %) in RS2 is at 0%, 0.025%, 0.25%.
Dilution RS4 in final measurement liquid RS2, be adjusted to acetate buffer solution (pH5.0) concentration be 300mM,
FeSO4Concentration be 0.03mM.Therefore, the concentration of acetate buffer solution (pH5.0) is adjusted to 2000mM.In addition, by FeSO4's
Concentration is adjusted in the acetate buffer solution (pH5.0) as 0.2mM.
Measurement is carried out using measurement system A according to measurement process shown in Fig. 4.At this moment, measurement device 2 is set as can
Show the absorbance of 510nm.
That is, filling the chromogenic agents solution of specified amount respectively in the chromogenic agents solution tank 11, dilution liquid bath 14 of bracket 1
(9) and the acetate buffer solution.In addition, dispensing the 50 μ L of tert-butyl hydroperoxide solution to sample slot 10.Then, it will prop up
In the setting to measurement device 2 of frame 1, start to measure.Tip 17 is installed to front end by the ozzle 21 of measurement device 2, will dilute liquid bath
14 126 μ L of the acetate buffer solution is dispensed into dilution trap 13.Then, ozzle 21 draws the tert-butyl hydrogen of sample slot 10
Peroxide solutions dispense its 14 μ L to dilution trap 13.
Thereafter, ozzle 21 draw chromogenic agents solution tank 11 chromogenic agents solution (9), by its 150 μ L to survey light pond
12 dispensings.Then, ozzle 21 is drawn from dilution trap 13 by the tert-butyl hydroperoxide solution and the acetate buffer solution structure
At dilution sample RS3, by the dilution sample RS3 of 30 μ L to survey light pond 12 dispense.Ozzle 21 is by drawing or ejecting stirring
The liquid in light pond 12 is surveyed, mixed liquor is made to become measurement liquid RS2.Thereafter, temperature adjustment block 23 will survey light pond 12 with 37 DEG C and keep the temperature 5 points
Clock surveys the absorbance that light unit 22 measures 510nm.
It is similarly measured for comparing chromogenic agents solution (4).
Measurement result is shown in FIG. 11.In the graph, the longitudinal axis indicates the absorbance at 510nm, and horizontal axis indicates tert-butyl
The concentration of hydroperoxides.It is high at the absorbance of highly acid that p-methyl benzenesulfonic acid is added in chromogenic agents solution RS1, after keeping
With good reactivity.That is, knowing into chromogenic agents solution (9) color development examination compared with not at highly acid of strong acidic condition
Agent solution (4) compares, it is suppressed that background coloration, and the reactivity for aoxidizing chromogenic agents is also kept.
As described above, knowing that the rising for being able to suppress background according to the present invention has dissolved oxidation color development to transport/take care of
The chromogenic agents solution RS1 of reagent.
Claims (9)
1. a kind of for inhibiting to the background rising for taking care of the chromogenic agents solution occurred when chromogenic agents solution
Method, measurement of the chromogenic agents solution for the ingredient in sample, and dissolved with oxidation chromogenic agents,
The oxidation chromogenic agents are selected from compound or its salt represented by following formula,
Or
The method includes the hydrogen ion exponent of the chromogenic agents solution is adjusted to pH2.9 adjusting process below.
2. the method for claim 1, wherein
In the adjusting process, the hydrogen ion exponent of the chromogenic agents solution is adjusted to pH2.1 or less.
3. method according to claim 1 or 2, wherein
The chromogenic agents solution is for measuring oxidative stress degree.
4. a kind of chromogenic agents solution, for the measurement of the ingredient in sample, and dissolved with oxidation chromogenic agents, wherein
The oxidation chromogenic agents are selected from compound or its salt represented by following formula,
Or
The hydrogen ion exponent of the chromogenic agents solution is adjusted to pH2.9 or less.
5. chromogenic agents solution as claimed in claim 4, wherein
The hydrogen ion exponent of the chromogenic agents solution is adjusted to pH2.1 or less.
6. chromogenic agents solution as described in claim 4 or 5, wherein
The chromogenic agents solution is for measuring oxidative stress degree.
7. a kind of kit, the measurement for the ingredient in sample, wherein
The kit includes chromogenic agents solution described in any one of claim 4 to 6.
8. kit as claimed in claim 7, wherein
The kit also includes the dilution for diluting the chromogenic agents solution,
The dilution is adjusted to the defined hydrogen ion exponent suitable for color reaction by buffer, and the chromogenic agents are molten
The hydrogen ion exponent of liquid be configured to by be changing by the diluted dilution it is described as defined in hydrogen from
Subindex.
9. a kind of measurement device, the measurement for the ingredient in sample, wherein
The measurement device is measured using kit documented by claim 7 or 8.
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| JP2014062845 | 2014-03-26 | ||
| JP2014-062845 | 2014-03-26 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000095654A (en) * | 1998-09-28 | 2000-04-04 | Kimi Kasei Kk | Hair dye composition |
| US6203579B1 (en) * | 1997-04-24 | 2001-03-20 | Henkel Kommanditgesellschaft Auf Aktien | Use of 1-substituted isatins to dye fibers containing keratin |
| JP2006347604A (en) * | 2005-06-17 | 2006-12-28 | Fiamo:Kk | Mouth member, and contents storage container |
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| IT1274165B (en) | 1994-09-21 | 1997-07-15 | Diacron Srl | "REACTIVE FOR THE DETERMINATION OF FREE RADICALS" |
| JPH09133671A (en) * | 1995-09-05 | 1997-05-20 | Konica Corp | Method and kit for discriminating quantity of residual chlorine and kit for detecting oxide |
| US6355489B1 (en) * | 1997-03-21 | 2002-03-12 | Diacron S.R.L. | Method for the determination of oxygen-centered free radicals |
| US6180412B1 (en) * | 1999-03-26 | 2001-01-30 | Hach Company | Test for chlorine in water |
| JP3918976B2 (en) | 1999-12-22 | 2007-05-23 | 東洋紡績株式会社 | Preservation method of phenylenediamine derivative hydrochloride |
| FR2807648B1 (en) | 2000-04-12 | 2005-06-10 | Oreal | KERATIN FIBER OXIDATION DYEING COMPOSITION AND DYEING METHOD USING THE SAME |
| US20030138959A1 (en) * | 2002-01-17 | 2003-07-24 | Carter Jesse M. | Method of detecting oxidizing adulterants in urine |
| JP2003287541A (en) * | 2002-03-28 | 2003-10-10 | Gc Corp | Kit and method for testing cariogenic bacteria |
| CN1330773C (en) | 2002-05-21 | 2007-08-08 | 爱科来株式会社 | Method of preventing wrong color formation of N-(carboxymethylaminocarbonyl)-4,4 -bis(dimethylamino)diphenylamine sodium, reagent solution for the method, and measurement method employing the method |
| JP2005274564A (en) * | 2004-02-27 | 2005-10-06 | Suido Kiko Kaisha Ltd | Coloring reagent and concentration measuring method |
| JP2009257909A (en) * | 2008-04-16 | 2009-11-05 | Wismerll Co Ltd | Analyzer of degree of oxidation stress |
| JP6284756B2 (en) | 2012-12-25 | 2018-02-28 | アークレイ株式会社 | Reagent for measuring degree of oxidative stress and method for measuring degree of oxidative stress |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6203579B1 (en) * | 1997-04-24 | 2001-03-20 | Henkel Kommanditgesellschaft Auf Aktien | Use of 1-substituted isatins to dye fibers containing keratin |
| JP2000095654A (en) * | 1998-09-28 | 2000-04-04 | Kimi Kasei Kk | Hair dye composition |
| JP2006347604A (en) * | 2005-06-17 | 2006-12-28 | Fiamo:Kk | Mouth member, and contents storage container |
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| US10145765B2 (en) | 2018-12-04 |
| EP2923691B1 (en) | 2018-10-31 |
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| US20150276561A1 (en) | 2015-10-01 |
| JP2015194493A (en) | 2015-11-05 |
| EP2923691A1 (en) | 2015-09-30 |
| JP6533077B2 (en) | 2019-06-19 |
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